Figure 5. Loss of Rbf in Hub Cells Causes Ectopic Niche Formation.

(A–F) Single confocal sections through the testis apex of E132^ts > GFP RNAi control testes (A and D) or E132^ts > Rbf RNAi testes (B, C, E, and F). RNAi was induced for 14 days at 29°C. (A–C) Testes immunostained with Fas III (hub; membranous green), PH3 (mitotic cells; nuclear green), and DAPI (nuclei; blue). Control testis (A) contains only a single hub (white outline), while those testes expressing either Rbf RNAi A (B) or Rbf RNAi B (C) in hub cells contain multiple hubs. (D–F) Testes immunostained with Armadillo (hub; membranous white), 1B1 (fusome; white), Zfh1 (CySCs; green), Vasa (germ cells; red), and DAPI (nuclei; blue).

(D–D^ʹʹ) Control testis with a single hub surrounded by both CySCs (yellow arrows) and GSCs represented by red cells with a dot fusome (yellow arrowhead). Merged (D), Zfh1 only (D^ʹ), and 1B1 and Arm only (D^ʹʹ) channels are shown.

(E–F^ʹʹ) Testes with Rbf knocked down in the hub using two independent RNAi lines denoted A (E–E^ʹʹ) and B (F–F^ʹʹ) contain multiple hubs (white outlines) scattered throughout the testis apex, each with CySCs (yellow arrows) and GSCs (yellow arrowheads). Merged (E and F), Zfh1 only (E^ʹ and F^ʹ), and 1B1 and Arm only (E^ʹʹand F^ʹʹ) channels are shown.

Scale bars represent 20 μm. See also Figure S5 and Video S4.